MATERIALS AND METHODS

Materials: Kaliapsis sp. sponge was collected on 15 October 2004 in the sea around Menjangan island, West Bali National Park, 20 m under sea level.

General instrument: Infrared spectrophotometer (IR) (FTIR 8201 PC Shimadzu, Ultraviolet Spectrometer (UV) (Milton Roy 3000), mass spectrometer EIMS (Electron Impact Mass Spectroscopy) of INCOS 50 (Finigan MT). Spektrometer Resonance Magnetic Nuclear (NMR) 500 MHz (Jeol), operating at radiofrequency of 500 MHz.

Isolation procedure: The bioactivity guided extraction, fractionation and isolation of the active isolate were conducted based on standard procedure as reported previously (Setyowati et al., 2007; Houssen and Jaspars, 2005).

Cytotoxicity evaluation: The cytotoxic evaluation was performed following an established standard procedure (Doyle and Griffiths, 1998).

RESULTS AND DISCUSSION

Isolate 1, transparent colorless needle crystals, MP (uncorrected) 132-134°C. The isolate was soluble in methanol and DMSO but water insoluble. TLC detection on Cerium (IV) sulfate showed brown color after 110°C heating for 10 min.

Isolate 1 showed infra red absorption at v 3500-3200 cm^-1 indicating the hydroxide (OH stretching) or amide (NH stretching) functional groups (Silverstein and Webster, 1998; Jenie et al., 2006). The isolate contained aliphatic alcohol as shown on the infra red absorption of aliphatic alcohol v 1200 and 1000 cm^-1. The absorption at v 2931.6 and 2839 cm^-1 showed the methyl and methylene functional groups. It was supported also by the infra red absorption at v 1477.4, 1434.9 and 1400.2 cm^-1. The absorption at v 1662.5 cm^-1 was the characteristics of carbonyl (-C = O) functional group of an amide and the absorption of v 1272.9 cm^-1 was due to the occurrence of ether linkage –C-O-C-. Therefore, the isolate 1 contained -NH-CO-H, -CH₃, CH₂, R-OH and –C-O-C functional groups.

The ¹H-NMR spectra showed that the isolate 1 (Fig. 1) showed the chemical shift of methyl (s), at δ_H 1.8 ppm (3H), two metilen protons at δ_H 3.8 and 2.4 ppm and one methin proton of secondary alcohol at δ_H 3.9 ppm.

From the spectra, it was clear the occurrence of proton at δ_H 11 ppm for amide proton (-CONH).

The ¹H-¹H Cosy spectrum (Fig. 2), showed the proton-proton correlations of isolate 1. This spectrum confirmed the proton chemical shifts (δ_H) ppm of 7.8 (s), 1.8 (s), 6.4 (t, J = 1.35 Hz), 2.4 (m), 4.4 (m), 3.9 (q, J = 7.3 Hz) and 3.8 (dd, J = 6.1, 7.3 Hz), assigned for protons at C-6, 7, 2’, 3’, 4’, 5’ and 6’, respectively.

Fig. 1:	The 1H-NMR spectra of isolate 1 in CD3OD

Fig. 2:	1H-1H COSY spectra of isolate 1

Figure 3, showed the ¹³C-NMR spectra indicating that isolate 1 had 10 carbon atoms. Two carbons was in a very downfield position, δ_C 166.5 and 152.5 ppm were assigned as the carbonyl carbons. The methyl peak was showed at δ_C 12.5 ppm. The APT (Attached Proton Test) showed the number of -CH₃, -CH₂, -CH and -C (quaternary carbon) of isolate 1 and this isolate consisted of one methyl (-CH₃), two methylenes (-CH₂) and five methines (-CH) and two quaternary carbons. Table 1 showed the chemical shifts assignment of the ¹³C signals.

Correlation of protons and carbon signals as showed at Table 2, were confirmed by the HMQC (Heteronuclear Multiple Quantum Coherence) spectrum. Long-range proton-carbon correlation was judged by the HMBC (Heteronuclear Multiple Bond Coherence) spectrum.

Table 1:	The 13C-NMR assignment of isolate 1 in CD3OD

This spectrum showed the correlation of CH₃ correlated to CO (C-4), H-6 to (C-2’), OH to C-4’. Figure 4 and Table 2, showed long-range correlation of ¹H-¹³C correlation of isolate 1 resulted from the HMBC spectrum.

Fig. 3:	13C-NMR and APT of isolate 1 in CD3OD

Fig. 4:	HMBC long-range correlation of isolate 1

The mass spectrum (EI-MS) (Fig. 5), showed that the isolate 1 had molecular weight of 242 for chemical formula of C₁₀H₁₄O₅N₂ based on its molecular ion peak of m/z 242.

Table 2:	HMBC long-range correlation of isolate 1

The EI-MS spectrum of isolate 1 showed the ion fragments at m/z 242 (3%), 207 (3%), 150 (3%), 126 (41%), 117 (100%, base peak), 99 (32%), 97 (11%), 73 (47%), 69 (18%), 43 (49%), 41 (15%). Figure 6 showed the structure of isolate 1 and Fig. 7 showed fragmentation pattern of isolate 1.

Cytotoxicity evaluation showed that isolate 1 inhibit the growth of HeLa, Raji, Myeloma and T47D cells in vitro. The isolate 1 decreased the percentage of living cells of HeLa (Fig. 8a), Raji (Fig. 8b), Myeloma (Fig. 8c) and T47D (Fig. 8d). It was likely a dose dependent since the more extracts in the cells, the more decreasing of the percentage of living cells.

Fig. 5:	EI-MS spectrum of isolate 1

Fig. 6:	Structure of isolate 1, 1-(tetrahydro-4-hydroxy-5-(hydroxymethyl)furan-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione

Fig. 7:	Splitting of fragmentation pattern of isolate 1 isolated from Kaliapsis sp. sponge

Fig. 8a:	Percentage of living HeLa cells vs. isolate 1 concentration

Fig. 8b:	Percentage of living Raji cells vs. isolate 1 concentration

Fig. 8c:	Percentage of living Myeloma cells vs. isolate 1 concentration

Fig. 8d:	Percentage of living T47D cells vs. isolate 1 concentration

It was showed that the cell growth for HeLa, Myeloma and Raji cells, in media control being increased up to 72 h however, for T47D cells being increased up to 48 h, then showed the cell death starting fro 72 h. It was likely that the nutrition in the media was not sufficient for cell growth. The IC₅₀ in vitro cytotoxicity of isolate 1 was 0.18, 7.9, 6.9 and 5.8 μg mL^-1 on Myeloma, T47D, HeLa and Raji cells, respectively.

ACKNOWLEDGMENTS

The author (EPS) would like to acknowledge the funding support from Indonesian Ministry of National Education (Hibah Bersaing Grant No. XV/1/2007). Mass spectra and NMR were provided by LIPI Research Center for Chemistry Serpong Indonesia. The staff is acknowledged.